Figure 2.
The HES4 transcription factor is a direct transcriptional target of NOTCH1 dimers. (A) Heat map and hierarchical clustering based on normalized values of the gene expression RNA-seq data. Human CB cells with N-only, N+L, or N+LTB viruses, expanded in vitro for up to 10 days for N-only and up to 15 days for the other 2 cell conditions, and followed by GFP+Cherry+ FACS before RNA isolation and sequencing. The differentially expressed genes, scaled to mean = 0 and standard deviation (SD) = 1, are represented (adjusted P value ≤.1 and logFC >0.5). The highlighted genes have a documented sequence-paired binding site (SPS) in the promoter region. (B) Lollipop plots of the Hallmark GeneSets found to be enriched in human NOTCH1-ΔE (wt)- or NOTCH1-ΔE-R1984A (R1984A)-transduced CB cells for each condition (N-only, N+L, or N+LTB) by Gene Set Enrichment Analysis (GSEA). (C) Volcano plot of differentially expressed genes (adjusted P value <.05) in human NOTCH1-ΔE (wt)- vs NOTCH1-ΔE-R1984A (R1984A)-transduced cell subsets for each condition (N-only, N+L or N+LTB). (D) Venn diagram showing the up- and downregulated genes with a recognized NOTcH1 SPS in the promoter region in human NOTCH1-ΔE (wt)-transduced CB cells in comparison with NOTCH1-ΔE-R1984A (R1984A)-transduced cell subsets that were identified in the RNA-seq data sets and shared across all 3 conditions (N-only, N+L, and N+LTB). (E) HES4 messenger RNA (mRNA) expression level in human NOTCH1-ΔE (wt) or NOTCH1-ΔE-R1984A (R1984A) transduced CB cells for each condition (N-only, N+L, or N+LTB) as reported in panel A. The transduced cells were sorted using FACS before performing the TaqMan reverse transcriptase-digital droplet polymerase chain reaction assay. The graphs report the result of 3 independent experiments performed in triplicate. ∗∗∗P < .001 (Student t test). FDR, false discovery rate; Max, maximum; Min, minimum; NES, normalized enrichment score.

The HES4 transcription factor is a direct transcriptional target of NOTCH1 dimers. (A) Heat map and hierarchical clustering based on normalized values of the gene expression RNA-seq data. Human CB cells with N-only, N+L, or N+LTB viruses, expanded in vitro for up to 10 days for N-only and up to 15 days for the other 2 cell conditions, and followed by GFP+Cherry+ FACS before RNA isolation and sequencing. The differentially expressed genes, scaled to mean = 0 and standard deviation (SD) = 1, are represented (adjusted P value ≤.1 and logFC >0.5). The highlighted genes have a documented sequence-paired binding site (SPS) in the promoter region. (B) Lollipop plots of the Hallmark GeneSets found to be enriched in human NOTCH1-ΔE (wt)- or NOTCH1-ΔE-R1984A (R1984A)-transduced CB cells for each condition (N-only, N+L, or N+LTB) by Gene Set Enrichment Analysis (GSEA). (C) Volcano plot of differentially expressed genes (adjusted P value <.05) in human NOTCH1-ΔE (wt)- vs NOTCH1-ΔE-R1984A (R1984A)-transduced cell subsets for each condition (N-only, N+L or N+LTB). (D) Venn diagram showing the up- and downregulated genes with a recognized NOTcH1 SPS in the promoter region in human NOTCH1-ΔE (wt)-transduced CB cells in comparison with NOTCH1-ΔE-R1984A (R1984A)-transduced cell subsets that were identified in the RNA-seq data sets and shared across all 3 conditions (N-only, N+L, and N+LTB). (E) HES4 messenger RNA (mRNA) expression level in human NOTCH1-ΔE (wt) or NOTCH1-ΔE-R1984A (R1984A) transduced CB cells for each condition (N-only, N+L, or N+LTB) as reported in panel A. The transduced cells were sorted using FACS before performing the TaqMan reverse transcriptase-digital droplet polymerase chain reaction assay. The graphs report the result of 3 independent experiments performed in triplicate. ∗∗∗P < .001 (Student t test). FDR, false discovery rate; Max, maximum; Min, minimum; NES, normalized enrichment score.

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